Lymphocyte activation markers predict the therapeutic response to immune checkpoint inhibitors: A case–control study

Abstract Background Reports on changes in circulating immune cells induced by immune checkpoint inhibitors (ICIs) and their association with therapeutic responses are limited. This study aimed to analyze the interaction between ICIs and circulating blood cells in real‐world practice and identify biomarkers associated with therapeutic efficacy. Materials and Methods Patients with non‐small cell lung cancer who received nivolumab, pembrolizumab, or atezolizumab monotherapy were eligible. Blood samples were collected before and 7–21, and 22–42 days after treatment, and changes in the number and characteristics of neutrophils and lymphocytes in the peripheral blood were analyzed. The efficacy of ICIs was evaluated based on the best overall response up to 3 months after treatment. The patients were divided into the following groups: patients with non‐progressive disease (non‐PD) and those with progressive disease (PD). Blood immunological factors related to treatment response were analyzed. Results There were 16 and 27 patients in the non‐PD and PD groups, respectively. The non‐PD group had significantly lower pretreatment CD56+ cell (CD56+ IFN‐γ+ cell), and neutrophil counts at 7–21 days after ICI administration than the PD group. Additionally, there was a significant increase in CD8+ IFN‐γ+, and CD8+CD28+ IFN‐γ+ T‐cell counts 7–21 days after ICI administration in the non‐PD group. CD8+CD28+IFN‐γ+ T‐cell count greater than 94.6 cells/μL at 7–21 days after ICI administration was highly predictive with an AUC greater than 0.8 in the ROC curve analysis. Conclusions Predictors of disease progression include a low activated CD56+ cell count, no increase in the neutrophil count before ICI therapy, and an increase of activated CD8+ cell count early after ICI administration. The findings will contribute to selecting patients with a favorable therapeutic response and early prediction of therapeutic response to ICI.


Materials and Methods:
Patients with non-small cell lung cancer who received nivolumab, pembrolizumab, or atezolizumab monotherapy were eligible.Blood samples were collected before and 7-21, and 22-42 days after treatment, and changes in the number and characteristics of neutrophils and lymphocytes in the peripheral blood were analyzed.The efficacy of ICIs was evaluated based on the best overall response up to 3 months after treatment.The patients were divided into the following groups: patients with non-progressive disease (non-PD) and those with progressive disease (PD).Blood immunological factors related to treatment response were analyzed.
Results: There were 16 and 27 patients in the non-PD and PD groups, respectively.The non-PD group had significantly lower pretreatment CD56 + cell (CD56 + IFN-γ + cell), and neutrophil counts at 7-21 days after ICI administration than the PD group.Additionally, there was a significant increase in CD8 + IFN-γ + , and CD8 + CD28 + IFN-γ + T-cell counts 7-21 days after ICI administration in the non-PD group.CD8 + CD28 + IFN-γ + T-cell count greater than 94.6 cells/μL at 7-21 days after ICI administration was highly predictive with an AUC greater than 0.8 in the ROC curve analysis.
Conclusions: Predictors of disease progression include a low activated CD56 + cell count, no increase in the neutrophil count before ICI therapy, and an increase of activated CD8 + cell count early after ICI administration.The findings will contribute to selecting patients with a favorable therapeutic response and early prediction of therapeutic response to ICI.

| INTRODUCTION
Treatment with antibodies against programmed deathligand 1 (PD-L1) or programmed cell death 1 protein (PD-1), or immune checkpoint inhibitors (ICIs) induces unprecedented tumor responses in various cancer types. 1 In a clinical trial comparing the efficacy of nivolumab, an ICI, with that of docetaxel as a second-line treatment in patients with non-small cell lung cancer (NSCLC), 8% of patients showed no progression at 1 year with docetaxel compared with 19% of patients who showed no progression with nivolumab, indicating that some patients continued to respond to treatment for a long period. 2owever, only a limited number of patients can sustain the therapeutic effect over a long period.In addition, as ICIs are expensive, high medical costs become a problem with an increased number of patients using these drugs.Therefore, the characterization of tumor microenvironmental factors and patient immune function at baseline and early during treatment to identify patients who will respond to therapy is a research priority in cancer care.
4][5] Recently, PD-L1-expressing CD11b + myeloid cells in the systemic circulation have also been reported to be associated with clinical response. 6However, a study in a mouse model in which PD-L1 was knocked out from tumor cells revealed no change in the anti-tumor effect of the anti-PD-L1 antibody, indicating that PD-L1 expression on tumors is not essential for PD-1 to suppress T-cell function. 7Moreover, since some patients with PD-L1-negative tumors show therapeutic responses, host immune function plays a major role in the therapeutic efficacy of ICIs. 8,9 characteristic of cancer cells with a good response to ICIs is a high frequency of genetic mutations.Improved overall survival with cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitor in patients with melanoma harboring more than 100 mutations and improved median progression-free survival in patients with lung cancer treated with pembrolizumab with a high mutation burden have been reported. 10,11In clinical practice, nivolumab and pembrolizumab have been approved for solid tumors with a high frequency of microsatellite instability (MSI-High). 12,13A study using whole-exome sequencing also revealed that 1782 somatic mutations on an average per tumor in mismatch repair-deficient tumors, compared with 73 somatic mutations in mismatch repair-proficient tumors, was associated with prolonged progression-free survival. 14dditionally, it has been reported that tumor mutation burden (TMB) is significantly higher in responders than in non-responders, and TMB > 23.1 Mut/Mb (TMB-high) is associated with a survival benefit compared with TMB ≤23.1 Mut/Mb (TMB-low or TMB-intermediate) in patients with metastasized or unresectable melanoma. 15egarding patient immune function, patients with high CD8 + T-cell counts at invasive margins of the tumor, 16 high peripheral blood lymphocyte counts, and low neutrophil counts 17 have a better prognosis, and the intestinal microbiota influences the response to treatment. 18,19CD8 + T-cell signaling (TCR) induce PD-1 expression in the cell surface, and when PD-l binds to its ligands (PD-L1 or PDL2), it inhibits TCR/ CD28 signaling and T-cell activation.Blockade of the PD-1 pathway reinvigorates exhausted CD8 + T cells and can restore antitumor immune response. 20Furthermore, PD-1, whose expression on CD8 + T cells increases following ICI administration, and CD28, a co-stimulatory molecule that enhances T-cell activity, are also attracting attention as candidate biomarkers for good clinical outcomes. 20,21However, there are only a few reports on CD28 expression changes by ICIs and their association with therapeutic response.NK cells, known as CD56 + cells, are type of cytotoxic lymphocyte that is critical to the innate immune system and account for 5%-20% of all circulating lymphocytes in humans.The role of NK cells is analogous to that of cytotoxic T cells in the adaptive immune response, however, there has been little investigation on the contribution of NK cells to the therapeutic effects of ICI.
This exploratory prospective case-control study was conducted in patients with NSCLC with the hope of extending the hematological molecular markers that are expected to predict therapeutic response to ICI to clinical applications.The results will contribute significance to selecting patients with a favorable therapeutic response to ICI.

| Patient selection, sample collection, and measurements
This single-center prospective study at Mie University Hospital involved patients with unresectable advanced or recurrent NSCLC at stage III or IV.The patients were biomarker, case-control study, immune checkpoint inhibitor, lymphocyte, non-small cell lung cancer 20 years or older, had a performance status (PS) of 0-1, and were scheduled to receive one of the following ICIs as a monotherapy: nivolumab (OPDIVO I.V. infusion; Ono Pharmaceutical Co., Ltd., Osaka, Japan), pembrolizumab (KEYTRUDA injection; Merck & Co., Inc., Rahway, NJ), or atezolizumab (TECENTRIQ for intravenous infusion; Chugai Pharmaceutical Co., Ltd., Tokyo, Japan).Patients were excluded if they required systemic treatment with steroids (prednisolone equivalent >10 mg/day) or other immunosuppressive agents within 14 days of enrollment or had concomitant autoimmune diseases.Cases in which treatment was discontinued within 2 weeks of ICI administration were also excluded.The PD-L1 expression level was evaluated using the tumor proportion score, measured using PD-L1 IHC 22C3 pharmDx, Dako (Agilent Technologies Japan, Ltd., Tokyo, Japan).
This study was approved by the Ethics Committee of Mie University (approval no.3062), and written informed consent was obtained from each participant between November 2017 and August 2020.The target number of participants was set at 20 for each of the non-PD and PD groups, with a ratio of 1:1 for each group and 10% would meet the exclusion criteria, for a total of 50 participants.

| Pharmacodynamic analysis and statistical analysis
This study evaluated the pharmacodynamic changes of immune biomarkers in blood NK and CD8 + T cells and their association with response to therapy with ICs at 3 months (Response Evaluation Criteria in Solid Tumors criteria version 1.1).Patients were divided into PD and non-PD groups 3 months after initiating ICIs.Hematological immune biomarkers were compared between the groups using the non-parametric Mann-Whitney U-test for numerical variables and Fisher's exact test for categorical variables.All tests of comparison between the two groups in this study showed the results of the exact p-value, and the results of the approximate p-value were not used.Receiver operating characteristic (ROC) curve analysis was performed to estimate the sensitivity, specificity, accuracy, and cutoff values of several hematological immune biomarkers and their association with the primary endpoint.Statistical analyses were performed using GraphPad Prism (version 9.3.1;GraphPad Software, San Diego, CA).Two-sided p-values were reported, and the results were considered significant at p < 0.05.

| Enrolment of patients
The study flow is presented in Figure 1.Forty-four patients were enrolled in this study, and one patient discontinued ICI within 2 weeks after initiation.A total of 43 patients (29 men and 14 women) were eligible for analysis.The median age at entry was 71 years (youngest, 41 years; oldest, 84 years).The patients' demographics of PD (n = 27) or non-PD (n = 16) groups are shown in Table 1.The body weight of non-PD group patients was higher than that of PD group patients.Furthermore, the PD group had higher EGFR mutations in the tumor.However, there were no significant differences between groups regarding age, sex, carcinoma, clinical stage, PD-L1 expression, type of ICI used, and lines of ICI therapy.

| Comparison of the hematological immune biomarkers between the PD and non-PD groups
There was no significant difference in lymphocyte count between the PD and non-PD groups before ICI ≥ Third line 8 Note: PD-L1 expression level was evaluated using tumor proportion score, which was measured using PD-L1 IHC 22C3 pharmDx, Dako (Agilent Technologies Japan, Ltd., Tokyo, Japan).
Abbreviations: ICI, immune checkpoint inhibitor; PD, progressive disease.administration and 7-21 and 22-42 days after administration (Table 2).For all patients, blood sampling timing on Days 7-21 were the timing after the first ICI administration.Regarding neutrophil count, there was no significant difference between the PD and non-PD groups before ICI administration, whereas there was a significant increase at 7-21 days post-administration in the PD group compared with that in the non-PD group.At 22-42 days post-administration, there was no significant difference in neutrophil count between the groups.Lymphocyte counts and neutrophil/lymphocyte ratios did not differ between the two groups before and after ICI administration.Typical flow cytometric quantification of the percentage of lymphocyte subsets is shown in Figure 2. Changes in CD56 + cell (NK cell) and CD8 + T cell counts in the non-PD and PD groups at 3 months after ICI administration are shown in Table 3. Concerning the number of CD56 + and IFN-γ + CD56 cells before ICI administration, the PD group had higher counts than the non-PD group (p < 0.0129 and p < 0.0422).On the contrary, there was no significant difference between the groups in CD56 + cell count at 7-21 and 22-42 days after ICI administration.Regarding CD8 + T cell count 7-21 days after ICI administration, the number of IFNγ + cells was significantly higher in the non-PD group than in the PD group (p = 0.0478).Similarly, the number of CD28 + cells tended to be higher in the non-PD group than in the PD group (p = 0.0722).Furthermore, as shown in Table 4, the number of CD8 + CD28 + IFN-γ + T cells 7-21 days after ICI administration and the increase in their count from baseline in the non-PD group were significantly higher than those in the PD group (p = 0.0006 and p = 0.0025).Contrarily, there was no significant difference in the CD8 + T cell markers between the groups before and 22-42 days after ICI administration.As for ICI binding to CD8 + T cells, there was no difference between the groups before and after ICI administration.
In summary, the non-PD group, which had a better treatment outcome, had lower NK cell activity before ICI administration, and no increase in neutrophil counts and CD8 cell activation with CD28 and IFN-γ expressions at 7-21 days after ICI administration.Figure 3 3.3 | ROC curve analysis predicting non-PD outcome for hematological immune biomarkers A ROC curve analysis was performed for CD56 + and CD8 + cell-related factors.The analysis showed significant differences between the PD and non-PD groups.Regarding neutrophil count 7-21 days after ICI administration, the cutoff value predicting non-PD was 5315 cells/ μL.The sensitivity, specificity, accuracy, and AUC were 100%, 51.9%, 69.8%, and 0.6944, respectively (p = 0.0348) (Figure 4).

| DISCUSSION
This study evaluated the relationship between hematological immune biomarkers in the blood and the clinical outcome of non-PD following ICI administration.The body weight of patients in the non-PD group was higher than that of patients in the PD group, suggesting a better nutritional status.The non-PD group also had a F I G U R E 2 Flow cytometry quantification of the proportion of lymphocyte subsets for CD3 + peripheral blood mononuclear cells (A) and CD3 − peripheral blood mononuclear cells (B).lower percentage of EGFR mutations in the tumors than the PD group, implying that the tumors tended to be less proliferative.Despite these background differences, the hematological features that facilitated the non-PD outcome included a low CD56 + cell count (CD56 + IFN-γ + cell count) before ICI administration, no increase in neutrophil count, and an increase in the number of activated CD8 + cells 7-21 days after ICI administration.To the best of our knowledge, this is the first study to suggest that the CD28 + IFN-γ + T cell count and its increase from baseline may be good indicators of increased activated CD8 + cell count and may be associated with good therapeutic outcomes following ICI administration.In particular, CD8 + CD28 + IFN-γ + T cell count greater than 94.6 cells/μL 7-21 days after ICI administration was highly predictive with an AUC greater than 0.8 in the ROC curve analysis, which may be useful in clinical practice for early prediction of therapeutic response to ICI.
CD28, a co-stimulatory molecule for T cell activation, is essential for T cells to recognize dendritic cells.Previous studies have shown that CD28 is preferentially dephosphorylated in response to PD-1 activation by PD-L1 in an intact cell system and that PD-1 suppresses T cell function mainly by inactivating CD28 signaling.These findings indicate that co-stimulatory pathways play key roles in regulating effector T-cell function and responses to ICI administration. 21Moreover, PD-1 antibodies activate the CD28/B7 pathway to rescue the depletion of CD8 + T cells and then achieve anti-tumor effects. 21,23A clinical trial that examined biomarkers predictive of ICI treatment response showed that ICI responders had higher baseline exosomal CD28 expression than non-responders and that the median progression-free survival of the high-CD28-expressing group was longer than that of the low-CD28-expressing group. 24In the current study, there was no difference in the number of activated CD8 + T cells in the bloodstream before ICI administration between the PD and non-PD groups, whereas the number of activated CD8 + T cells increased in the non-PD group early after ICI administration (Days 7-21).This phenomenon can be explained by the fact that ICI responders quickly respond to ICIs and that CD8 + T cells are activated through increased production of IFN-γ and CD28.An ex-vivo functional assay to determine CD8 + T cell activation after PD-1 blockade in circulating peripheral blood showed increased intracellular IFN-γ + cells. 25This study confirmed that the non-PD group has an increase in CD8 + IFN-γ + cell count early after ICI administration.Ottonello et al. also showed that in 74 patients with advanced NSCLC treated with nivolumab, patients with longer overall survival had lower NK cell counts at baseline. 26his finding supports our result that the non-PD group had a lower CD56 + cell count (CD56 + IFN-γ + cell count) than the PD group before ICI administration.However, a phase II study on pembrolizumab involving patients with advanced NSCLC reported that a higher NK cell count at baseline and the first radiologic evaluation was associated with improved clinical outcomes. 27A prospective study of the circulating immune profile in patients with NSCLC receiving nivolumab also showed that at baseline, the clinical benefit group (n = 19) had a higher number of active NK and PD-1 + CD8 + cells than non-responders (n = 12). 28Moreover, a prospective exploratory study of 40 patients with small-cell lung cancer treated with chemotherapy and ICIs showed that disease progression correlated with a low NK cell count. 29These results indicate that the specific function of NK cells in immunotherapy and their potential as biomarkers for predicting response to ICIs need further investigation.
An elevated neutrophil/lymphocyte ratio indicates chronic inflammation and reflects the immune status of patients with different malignancies. 30Several studies have investigated the negative prognostic value of a high neutrophil/lymphocyte ratio in patients with NSCLC receiving immunotherapy.In addition, an analysis of clinical blood test data from 249 patients with NSCLC treated with ICIs revealed that a high neutrophil/lymphocyte ratio of 5 before and after ICI administration was associated with a low survival rate. 31Suh et al. also reported that a neutrophil/lymphocyte ratio ≥5 at 6 weeks post-ICI administration is associated with poor progression-free survival and overall survival. 32As mentioned earlier, a low neutrophil count (<4000/μL) is reportedly associated with lower response rates in ICI treatment. 17Taken together, these reports support our results showing that no increase in neutrophil count 7-21 days after ICI administration is associated with a good clinical outcome, although our data did not show a difference of the neutrophil/lymphocyte ratio between the non-PD and PD groups.Therefore, in addition to a neutrophil/lymphocyte ratio, an absence of an increase in neutrophil count after ICI administration could be a good predictor of the therapeutic effect of ICIs.
This study has some limitations.First, the findings of this pilot study are limited by the small number of patients enrolled.Second, this study focused on the early phase clinical outcome (initial 3 months) after ICI administration and did not perform a multivariate analysis of factors associated with non-PD because of the small sample size.However, this exploratory prospective case-control study provides solid clinical evidence with a large statistical difference in predicting response to ICIs, especially concerning the dynamics of CD8 + CD28 + IFN-γ + T cells after ICI administration.
To conclude, this exploratory study showed a relationship between peripheral blood immune cell reactivity and clinical response to ICIs in patients with NSCLC.We found that the lack of an increase in the number of neutrophils and the increase in circulating CD8 + CD28 + IFN-γ + T cells after ICI administration were associated with favorable clinical outcomes.The findings will contribute to selecting patients with a favorable therapeutic response and early prediction of therapeutic response to ICI and warrant further validation in larger studies in the future.These findings will be of interest to healthcare professionals involved in cancer treatment, immunotherapy, and personalized drug therapy.

F I G U R E 3 F I G U R E 4
Typical histograms of flow cytometric analysis of CD56 + IFN-γ + cells before ICI administration and CD8 + CD28 + cells, CD8 + IFN-γ + cells, and CD8 + CD28 + IFN-γ + cells approximately 10 days after ICI administration in a non-PD patient (Patient 13 day 11) (A) and a PD patient (Patient 12 day 10) (B).Histograms were represented in comparison to the respective isotype control.Receiver operator characteristic (ROC) curve analysis of the neutrophil count 7-21 days after ICI administration for predicting non-PD.
Demographic of non-PD and PD groups before ICI administration.
T A B L E 1 cells/μL.The sensitivity, Abbreviations: ICI, immune checkpoint inhibitor; PD, progressive disease.